Access to a library of structurally defined N-glycans is an urgent scientific need for glyco-arrays for rapid analysis of carbohydrate binding proteins (CBPs), development of diagnostics and therapeutics for many diseases. However, only a small number of N-glycans are currently commercially available, which hampered researches in Glycobiology. This application will chemo-enzymatically prepare a library of 80 N-glycans (hybrid-, linear and complex-type) and commercialize them. The long-term goal is to produce thousands of natural N-glycans for carbohydrate-based highthroughput assays. Firstly, essential N-glycan precursors will be prepared in gram scales via either chemical synthesis or isolation from chicken eggs yolk and white. These N-glycans will then be enzymatically elongated or digested to yield 80 different N-glycans with defined structures (1 mg), among which only 12 N-glycans are currently available from commercial sources. The N-glycans contain 5 to 11 monosaccharide residues. This process involves several well characterized glycosyltransferases, glycoside hydrolases and other enzymes. All the N-glycans will be purified to 98%, and fully characterized by NMR and MS.